Mechanobiology describes the relationship between a cell and its environment; how a cell can detect, measure and respond to the rigidity of its substrate and how these processes apply to larger biological systems.

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The detection of mechanical signals, and their integration into biochemical pathways, is integral to the cell’s ability to sense, measure and respond to its physical surroundings. Mechanosignl and enable communication between neighbouring cells. Learn More

Genome regulation encompasses all facets of gene expression, from the biochemical modifications of DNA, to the physical arrangement of chromosomes and the activity of the transcription machinery. Learn More

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Insights into disease etiology and progression, the two major aspects of pathogenesis, are paramount in the prevention, management and treatment of various diseases. While many people will be genetically predisposed to a given disease, the mechanical properties of the tissue or cellular environment can also contribute to disease progression or its onset.Learn More

How do forces affect actin remodeling at distant locations in a cell?Sruthi Jagannathan2018-05-31T14:00:32+08:30

Project Description

How do forces affect actin remodeling at distant locations in a cell?

Forces applied at the periphery of a cell causes actin remodeling around the nucleus, leading to the formation of a transient perinuclear actin rim. The formation of this actin structure is dependent on the force-induced release of Ca2+ ions in the cytoplasm, and is mediated by the formin family member, INF-2.

Figure: The schematic represents the long-acting effects of locally applied forces. a) local forces (100-200 nN) are applied at the cell periphery using an AFM tip. b) application of forces triggers the release of intracellular Ca2+ ions. c) Ca2+ signaling induces actin remodeling around the nucleus via inverted formin-2 (INF-2), leading to the assembly of a transient perinuclear actin rim.

Summary

Cells respond to the physical properties of their surroundings by remodeling their cytoskeleton, mainly actin filaments, into higher order structures that can propagate forces across the cell. Numerous studies have shown how forces affect actin remodeling directly at sites of force application. However, their effects on actin filaments at distant locations remained uncharacterized until recently.

This study reveals a previously unidentified, far-acting effect of forces on actin remodeling. By using a specially designed atomic force microscopy tip to apply forces of a magnitude between 100-200 nN at the cell periphery, the researchers noted the assembly of actin filaments around the nucleus into a transient high-order structure, which they called the perinuclear actin rim. The assembly of the perinuclear actin rim was dependent on force-induced release of Ca2+ in the cytoplasm, and was found to be mediated by the actin filament nucleation and elongation factor, inverted formin-2 (INF-2).

The perinuclear actin rim is associated with two main functions: relaying mechanical signals from the cytoplasm to the nucleus, and shielding the genome from any mechanical perturbations that could reach the nucleus.

Understanding the basics

The actin cytoskeleton is physically connected to the extracellular matrix (ECM) through multiprotein complexes known as focal adhesions. Interactions between cell-surface molecules such as integrins and the actin cytoskeleton are bidirectional, with focal adhesions forming links between them. The actin cytoskeleton and the focal adhesions function as mechanosensors: they convert the strength of the adhesions and the tensile forces along the actin cytoskeleton into biochemical signals that control cell behavior. The physico-chemical properties of the ECM, such as its rigidity, topography, and chemical composition, as well as stresses propagated via focal adhesions will influence the assembly and organization of the actin cytoskeleton.

Formins promote the elongation of pre-existing actin filaments by removing barbed end capping proteins and forming a sleeve around actin subunits. Formins are also capable of actin nucleation, which is the de novo assembly of actin filaments from actin monomers. Activated formins exist as dimers and form a donut-shaped complex around terminal actin subunits, orienting themselves toward the barbed (+) end of actin filaments. The FH2 domain facilitates binding to the growing filament; this requires removing capping proteins from the barbed ends and preventing re-capping in order to allow continued growth of actin filaments. Ena/VASP proteins support formin-mediated filament elongation by tethering the filaments near sites of active actin assembly. Each formin monomer then binds and captures profilin proteins through its FH1 domain, which are already bound to G-actin monomers. The monomers are then added to the growing actin filament.

Cytoskeletal filaments- actin filaments, intermediate filaments, and microtubules, bridge the nucleus to the plasma membrane, which in turn is anchored at sub-cellular sites to extracellular substrates via a plethora of proteins that form focal adhesions. The cytoskeletal filaments converge on the nucleus, where they bind directly to KASH domain proteins such as the nesprin proteins. These proteins localize on the outer nuclear membrane and are part of a larger complex known as the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex: a physical link that enables force transmission across the nuclear envelope to impact processes within the nucleus. Such transduction is possible as nesprin proteins are connected to the inner nuclear membrane through dimers of the SUN-domain adaptor proteins (Sad1p, UNC-84). These SUN proteins are in turn anchored to nuclear lamins, and chromatin, inside the nucleus. These links are emerging to be pivotal in various physiological processes including cell migration, and in the ability of cells to cope with mechanical stress.

The first step in the formation of a filopodium is the nucleation of actin filaments from G-actin monomers. This is facilitated by various proteins known as nucleators, and may occur via the ‘tip nucleation model’ or the ‘convergent nucleation model’… Read more…

How does the cytoskeleton influence nuclear morphology and positioning?steve2018-01-19T16:12:40+08:30

Work by Mazumder et al. ascertained the active involvement of cytoskeletal forces in determining nuclear morphology. Change in nuclear size upon perturbation of actomyosin and microtubules affirmed their roles in exerting tensile and compressive forces respectively on the nucleus, correlating with their functions in the cellular context , … Read more…

How does the cytoskeleton couple the plasma membrane to the nucleus?steve2018-01-19T16:24:16+08:30

Cytoskeletal filaments bridge the nucleus to the plasma membrane, which in turn is anchored at sub-cellular sites to extracellular substrates via a plethora of proteins that form focal adhesions (FAs). FAs are points of cross-talk between transmembrane integrin receptors and the cytoplasmic filaments and thus are key sites for both biochemical and mechanotransduction pathways… Read more…

How do actin filaments respond when external forces are applied to the cell?steve2017-12-18T11:05:53+08:30

Actin filaments and their associated focal adhesion complexes act as information handling machines or mechanosensors: they convert both the strength of the adhesion and the tensile forces along the linked network of actin filaments (and associated proteins) into biochemical signals that control actin extension and cell migration… Read more…

How do actin filaments respond to changes in the internal cellular forces?steve2017-12-18T11:03:52+08:30

The orientation of individual actin filaments in the cytoskeleton is a force-driven evolutionary process that contributes to the elastic behavior of the network and influences whether a filament will deform by compression, bending or extension. Cross-linked actin networks initially become more elastic under low force as a result of filament resistance to the direction of compression. As the force increases, individual filaments inherently resist being compressed and/or cross-linking proteins become more extended, which causes the cytoskeleton network to become more rigid; cell stiffening has also been correlated with actin recruitment… Read more…

What factors influence the protrusive force of actin filaments?steve2017-12-18T11:03:25+08:30

Factors that influence the concentration of free G-actin (e.g. profilin [40]) or ATP-binding and hydrolysis on actin will promote filament assembly and membrane protrusion. Furthermore, the microtubule and intermediate filament networks also play a key role in regulating the global deposition pattern of the actin filaments and therefore also influence membrane protrusion dynamics… Read more…

Actin filaments are initiated with their barbed ends oriented towards the plasma membrane, with ATP hydrolysis facilitating filament growth. Polymerization is favored towards the cell front and disassembly occurs more frequently at the rear. However, only a small fraction of the overall free energy of nucleotide hydrolysis is needed to modulate G-actin monomer binding. The remaining energy is translated into a protrusive force that deforms the plasma membrane in a particular direction… Read more…

The Study in Detail

Key Findings

The effects of external forces on actin remodeling at distant locations inside the cell was assessed by applying forces (100-200 nN) at the periphery of NIH3T3 cells using a specially designed atomic force microscopy (AFM) tip. A transient actin structure assembled around the nucleus in response to forces, which was referred to as the perinuclear actin rim.

Filamentous actin (F-actin) levels around the nucleus reached a maximum at 30 seconds following the application of forces and later returned to original levels within 2 minutes. F-actin levels nearer to the cell membrane showed a corresponding decrease with the increasing levels at the perinuclear region.

There was a sudden release of Ca2+ ions from intracellular calcium stores in response to forces and this intracellular Ca2+ burst preceded perinuclear actin rim formation. Once the Ca2+ in the cytoplasm returned to basal levels, the actin rim around the nucleus also disappeared.

When intracellular Ca2+ was depleted by EGTA treatment, the perinuclear actin rim failed to form; alternately treatment of NIH3T3 cells with a calcium ionophore (induces intracellular ca2+ release) was sufficient to trigger the formation of the perinuclear actin rim, even in the absence of mechanical forces. These findings suggested an indispensable role of Ca2+ signaling in the actin remodeling process.

The formation of the perinuclear actin rim was independent of integrin signaling, which was confirmed by observations of actin rim formation in cells grown on poly-L-lysine substrates (i.e. without integrin ligands) or in cells treated with an FAK (integrin signaling component) inhibitor.

Perinuclear actin rim formation in response to force/ionophore induced-Ca2+ stimulation was not prevented when cells were treated with inhibitors for actin regulators such as Arp2/3, Myosin-II, Cofilin, and Rho GTPase. This suggested that a different molecular pathway was responsible for the actin remodeling event.

However, when inverted formin-2 (INF-2) was knocked out, the actin rim failed to form. INF-2 is an actin filament nucleation and elongation factor belonging to the formin family. This suggests crucial role for this protein in perinuclear actin polymerization. Moreover, INF-2 mediated actin polymerization was dependent on Ca2+ stimulation, since overexpression of INF-2 in the absence of Ca2+ release did not induce actin rim formation.

Methods and Controls used in the study

An atomic force microscopy (AFM) tip attached to a 4.5 micrometer polystyrene bead that was controlled by a micromanipulator was used to apply forces at the periphery of NIH3T3 cells.

Confocal microscopy was used for live imaging of cells in order to quantify the response of cells to application of local forces.

Applications and Future Directions

The study provides insights into the physiological relevance of force/Ca2+-induced perinuclear actin remodeling: on one hand, it aids in the relay of mechanical signals from the cytoplasm to the nucleus by facilitating the nuclear transport of transcription factors; on the other hand, it functions as a physical barrier or a shield that protects the integrity of the genome during mechano-chemical instabilities within the cell.

By linking INF-2 mediated perinuclear actin polymerization with genome function, the study provides clues regarding defects in INF-2 and the occurrence of diseases such as focal and segmental glomerulosclerosis and Charcot-Marie Tooth disease.